Recent events such as the spread of swine flu and potential future detrimental mutations of influenza viruses point to an urgent need for a point-of-care influenza typing system;particularly because over 90% of hospitals and smaller clinical labs in the U.S. cannot perform DNA-based diagnostic analysis. To enable a quick response to a potential influenza outbreak, it is desirable to have a fast, accurate diagnostic method capable of simultaneously typing and subtyping influenza viruses. Innovative solutions are needed in sample preparation, DNA/RNA extraction, microfluidics, DNA amplification methods as well as instrument miniaturization to make bacterial and/or viral point-of-care molecular diagnostics a reality. This project leverages a feasibility of on-chip, real time rolling circle amplification of DNA on an electronic microarray platform demonstrated in Phase I with our efforts in miniaturization of DNA detection and development of platforms for integration with the sample preparation. The following innovative solutions form the basis of the proposed technology:(i) novel real-time fluorescence detection of (branched) rolling circle amplification of DNA on the electronic microarray platform;(ii) a disposable cartridge that incorporates a unique fluidics enabling magnetic bead DNA/RNA extraction, reverse transcription, DNA amplification and microarray detection;(iii) low cost, screen printed carbon electrodes-based microarray for electric field-assisted acceleration of DNA hybridization. The Phase II project specific aims will demonstrate the feasibility of the technology that will consist of: (1) Optimization of real-time rolling circle amplification (RCA) conditions on the carbon-based electronic microarray;(2) Development and optimization of influenza virus genotyping assay based on hemagglutinin (HA) and neuraminidase (N) typing;(3) Development of sample preparation chemistry for influenza typing assay and incorporation onto a disposable cartridge using magnetic bead based separation of RNA/DNA, reverse transcription and RCA amplification;(4) MDx system development and adaptation to influenza assay;and, (5) Validation of the influenza assay using spiked and clinical (archived) samples. PUBLIC HEALTH RELEVANCE: The recent spread of the H1N1 swine-origin influenza virus and potential detrimental mutations of influenza viruses point to an urgent need to develop point-of-care systems that will be capable of typing influenza viruses. Molecular diagnostics methods providing accurate typing of a virus will ensure a quick and appropriate response and enable improved management of patients and limit transmission of viruses. Standard techniques for viral typing are mostly based on PCR or real-time RT-PCR methods that are complex and require several hours to perform the analysis. This project brings a new real-time method for detection of DNA based on an extremely rapid rolling circle amplification of DNA/RNA targets on the electronic microarray platform. In the proposed project, the instrument and the assay will be fully developed and provide a low-cost, portable solution to diagnose influenza and/or other infectious diseases.